This invention relates to products and methods for increasing production of proteins in mammalian cells of lymphoid origin. More particularly, it relates to products and methods for increasing protein production by increasing the steady state level of translatably competent mRNA by decreasing the rate of intracellular degradation of the mRNA.
Protein production in most animal cells includes synthesis of enzymes, structural proteins, surface proteins, and numerous proteins having specialized functions such as lymphokines and hormones. Typically, relatively modest amounts of these proteins are produced. There are, however, types of animal cells which are capable of producing and secreting large amounts of proteins for systemic use in the animal body. Examples of the latter type of cells include cells of the circulatory system which produce globulins and fibrinogen, liver cells which produce serum albumin, and the beta cells of Islets of Langerhans which produce insulin. If the genetic mechanisms responsible for such high level expression could be exploited to produce lymphokines, antibodies, or other proteinaceous materials of interest, large supplies of valuable proteins could be made available.
Expression of endogenous DNA in eucaryotic cells involves transcription of the DNA into mRNA, subsequent migration of the mRNA to ribosomes followed by tRNA-mediated translation of the mRNA into proteins comprising the sequence of amino acids encoded by the mRNA between its start and stop signal codons.
As disclosed by Gillies et al in Cell, Vol. 33, pp. 717-728, July, 1983, and in co-pending U.S. application Ser. No. 592,231, filed Mar. 22, 1984, now U.S. Pat. No. 4,663,2 cellular enhancer elements play an important role in the high level expression of protein in specialized cells which produce large amounts of immunoglobulin. The enhancers appear to function by increasing the rate of DNA transcription into mRNA by endogenous mRNA polymerase. Increased concentrations of mRNA result in significant increases in the level of expression in such cells. The activity of such enhancer elements is independent of orientation and can be observed even when the sequence comprising the enhancer is located 10,000 base pairs or more away from the promoter for the gene encoding the protein. These cellular enhancers appear to be tissue specific, i.e., the activity of a cellular enhancer which functions in the endogenous genome of a lymphoid cell to increase production of a particular mRNA is greatly decreased or absent if the enhancer is incorporated in a vector used to transform non-lymphoid cells. However, a vector including the enhancer element, promotor, and a recombined gene encoding a desired protein, if transfected into a cell of the same type as that in which the enhancer naturally increases the transcription rate, can successfully transform the cell to express the recombined gene at high levels.
Eucaryotic DNA's comprise a sequence of bases beyond the stop signal, a portion of which serve as a signal to initiate addition of adenine residues (hereinafter poly A) 3' of the stop signal in the translated mRNA. The polyadenylation occurs mainly in the nucleus and is mediated by poly A Polymerase that adds one adenylic acid residue at a time. Poly A does not code for an amino acid sequence after the stop codon has terminated translation, but is thought to contribute to stabilization of mRNA's and to the efficiency of translation of the mRNA coding region into amino acids.
The poly A in the mRNA of mammalian cells lies in a sequence known as the 3'untranslatable (hereinafter 3'UT) end portion. The 3'UT typically extends from the termination codon for the translation product to the terminus of the poly A. The 3'UT regions of mammalian mRNA's typically have an area of homology, known as the AAUAAA hexanucleotide sequence. This sequence is thought to be the poly A addition signal. It often precedes by 11 to 30 bases the poly A addition site.
The function, if any, of the 3'UT and poly A region has been investigated recently by Soreq et al. (Proc. Natl. Acad. Sci. U.S.A. Vol. 78, No. 3, pps. 1741-1745, March, 1981); Zaret et al. (J. Mol. Biol., 1984 Vol. 176, pp. 107-135); Baralle (International Review of Cytology, Vol. 81, 1983, pp. 71-106); and Ross et al. (J. Mol Biol., 1983, Vol. 167, pp. 607-617).
Soreq et al. reported that removal of the poly A and approximately 100 adjacent residues from a first human fibroblast beta interferon mRNA did not alter the translational activity or the functional stability of this mRNA in oocytes, whereas the deletion of the poly A and approximately 200 adjacent residues did decrease its translational efficiency. The removal of approximately 200 poly A residues and 200 adjacent residues from a second beta interferon mRNA did not alter either the translational activity or functional stability of the mRNA in oocytes. These authors concluded that neither the poly A residues nor large segments of the 3' noncoding region are required for the maintenance of the functional stability of human beta interferon mRNAs in such oocytes.
Zaret et al studied the cyc 1-512 mutant of the yeast S. cerevisiae which contains a 38 base pair deletion in the 3' noncoding region of the CYC-1 gene which encodes iso - 1 - cytochrome c. They reported that different 3' noncoding sequences which arose by chromosomal rearrangement increased the stability of CYC-1 mRNA and have varying effects upon the mRNA translational efficiency.
In Baralle's publication, The Functional Significance of Leader and Trailer Sequences in Eucaryotic mRNAs, the author reported that there is "no obvious function for the 3' non coding region" and that "the occurrence of sizable deletions or insertions during evolution of these genes suggests that the particular sequences which comprise the 3' non coding region are not essential to mRNA function." Baralle reported however that poly A does have a role in promoting mRNA stability. For instance, it has been found that removal of the poly A segment from globin mRNA greatly decreases the half-life of the mRNA in oocytes. However, Baralle suggests that poly A is apparently not necessary for successful translation as has been demonstrated by studies wherein the poly A has been removed from the mRNA.
Ross and Pizarro studied the hypothesis that steady state levels of human beta and delta globin Proteins are determined in part by the intracellular stability of their respective messenger RNAs. They found that the rapid turnover of delta globin in mRNA accounts, at least in part, for the low level of delta globin mRNA in non nucleated peripheral blood reticulocytes, and speculated that the rate of mRNA decay may be determined by nucleotide sequence signals located in the 3' untranslated region. They observed that the 5' untranslated regions of beta and delta globin mRNAs are similar, but that their 3' untranslated regions differ significantly, and proposed that it should be possible to test the role of the 3' untranslated region in determining mRNA stability by comparing the half lives in transfected cells of chimeric mRNAs containing beta or delta 3' termini.
European Patent 0077689 discloses a method of gene manipulation wherein a yeast is transformed with a gene in which the 3'UT of a structural gene is added downstream from an exogenous gene. The transformant reveals a higher level of expression when the exogenous gene includes the 3'UT. In fact, studies revealed that the expression in the yeast carrying the plasmid with the region corresponding to the 3'UT as added is about 10-fold as compared with the yeast carrying the plasmid with no such region added.
It is an object of this invention to provide a product and process for efficient production of a desired protein including human tPA in certain types of animal cells. Another object is to provide vectors for transfecting animal cells to induce high level expression of a gene encoding a desired protein. Another object is to provide transformants which when cultured produce large amounts of protein for therapeutic, diagnostic, and related uses. Still another object of the invention is to provide methods and recombinant DNAs which promote mRNA stability in transfected cell lines by reducing the rate of intracellular mRNA decay, thereby increasing levels of expression.
These and other objects of the invention will be apparent to those skilled in the art from the following description and claims.